Abstract
A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to approximately 13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4-26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650-26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase alpha (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.
Highlights
Previous studies have focused on the helicase-primase and polymerase separately
Fraction of Primers Elongated by the DNA Polymerase in Coupled Activity—Primer utilization values were determined by comparing the total moles of product synthesized in the coupled assays to the total moles of RNA primers Ͼ7 nucleotides long synthesized in the primase assays under otherwise identical conditions
When assays contained NTPs and [␣-32P]dNTPs, radiolabeled products ϳ35– 40 nucleotides long were produced on the ssDNA template, 3Ј-d(T20GTCCT36)-5Ј (Table 1)
Summary
Reagents and Protein Purification—HSV-1 helicase-primase (UL8-UL5-UL52) and His-UL8 were expressed in baculovirus-. Primase-coupled Polymerase Activity Assay—Primase-coupled polymerase activity was measured in assays as described above, except that they contained 10 M [␣-32P]dNTPs, and the NTPs were unlabeled. Primer Length Assay—To determine the length of RNA primers elongated by the polymerase, coupled products were visualized by phosphorimagery, followed by gel extraction using double-distilled H2O and filtration through micro-spin cellulose acetate membranes (PerkinElmer Life Sciences) [26]. Fraction of Primers Elongated by the DNA Polymerase in Coupled Activity—Primer utilization values were determined by comparing the total moles of product synthesized in the coupled assays (measured using [␣-32P]dNTPs) to the total moles of RNA primers Ͼ7 nucleotides long synthesized in the primase assays (measured using [␣-32P]NTPs) under otherwise identical conditions. Fraction of Primers Transferred Intramolecularly—The fraction of primers transferred intramolecularly was determined by measuring the amount of coupled products formed in assays containing binase divided by the amount of coupled products formed in assays lacking binase
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